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Annu Rev Biophys. 2018 Mar 14. doi: 10.1146/annurev-biophys-070317-033123. [Epub ahead of print]

High-Resolution Hydroxyl Radical Protein Footprinting: Biophysics Tool for Drug Discovery.

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Center for Proteomics and Bioinformatics, and Department of Nutrition, Case Western Reserve University, Cleveland, Ohio 44106, USA; email:


Hydroxyl radical footprinting (HRF) of proteins with mass spectrometry (MS) is a widespread approach for assessing protein structure. Hydroxyl radicals react with a wide variety of protein side chains, and the ease with which radicals can be generated (by radiolysis or photolysis) has made the approach popular with many laboratories. As some side chains are less reactive and thus cannot be probed, additional specific and nonspecific labeling reagents have been introduced to extend the approach. At the same time, advances in liquid chromatography and MS approaches permit an examination of the labeling of individual residues, transforming the approach to high resolution. Lastly, advances in understanding of the chemistry of the approach have led to the determination of absolute protein topologies from HRF data. Overall, the technology can provide precise and accurate measures of side-chain solvent accessibility in a wide range of interesting and useful contexts for the study of protein structure and dynamics in both academia and industry. Expected final online publication date for the Annual Review of Biophysics Volume 47 is May 20, 2018. Please see for revised estimates.

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